Method and apparatus for liquid chromatography

ABSTRACT

In a method of filling a liquid chromatographic column with particulate separation medium, a liquid suspension of the particulate separation medium is introduced into one end of the column, wherein the column is closed at one end with a first filter, so that the particles will be retained by the filter while the liquid passes therethrough. After terminating the filling process, the other end of the column is closed with a second filter. According to the invention, the particle suspension is delivered via an inlet element which can be moved axially in relation to the end of the column an in which the second filter in mounted. During the filling process, the inlet element and the filter are held in a position in which the particle suspension is able to pass from the upper side of the filter and past its side-edge and into the interior of the column. After completion of the filling process, the inlet element is moved towards the end of the column, while maintaining liquid pressure, to a position in which by-passage of the filter is prevented, whereafter the inlet element is fixed in this position. The invention also relates to a column for carrying out the method.

The present invention relates to a column for liquid chromatography,which includes a column tube intended to be filled with a separationmedium, and inlet and outlet means. The invention also relates to amethod of filling such a column tube with separation medium.

In order for a chromatographic column to be able to separate biologicalsubstances effectively, it is usual to pack fine particles of separationmaterial as tightly and as uniformly as possible in the column tube.Filling of the column, or column packing as it is often referred to, isnormally effected by closing one end of the column with an outlet meanswhich includes a filter element, and pumping a liquid suspension of theparticles under pressure into the other end of the column. Whereas thepumped liquid is able to pass through the filter element essentiallyunobstructed, the particles are retained by the filter element, so as tobuild up a particle bed along the length of the tube. As the column tubeis filled, the particles are pressed out towards the wall of the tubeand the particle bed obtains a stable compaction state with theparticles well distributed, this state being maintained during the wholeof the filling process.

However, when the column tube has been filled with particles and pumpingof the liquid suspension is terminated to enable an inlet element to befitted to the filling-end of the tube, the stable restraining force inthe particle bed is partially lost, resulting in expansion of theparticle bed. Consequently, when the column tube is once again placedunder pressure, disturbing heterogeneities or irregularities are liableto occur in the particle bed, such as the formation of channels and deadvolumes.

Numerous attempts have been made to solve this problem. For instance,EP-A-0040663 suggests that the inlet element is provided with a springconstruction which will maintain the column bed under pressure andtherewith improve bed stability. A similar construction based on theeffect produced by a pressure plunger is described in DE-A-3021366 andU.S. Pat. No. 4,350,595. DE-A-3000475 describes a column having aflexible wall which stabilizes packing of the particles by radialcompression of the column packing. A column-filling device including apressure plunger which functions to compress the bed during the actualfilling process but which is then removed is described in U.S. Pat No.4,549,584. The construction that, at present, has been best establishedcommercially for improving bed stability is one in which the inletelement is provided with a hand-adjustable plunger arrangement or thelike, for instance as described in U.S. Pat. No. 4,737,292. A slightlydifferent concept is proposed in U.S. Pat. No. 4,732,687, whichdescribes the use of a perforated plate which is mounted in the upperpart of the column and through which a particle slurry can be introducedbut which is intended essentially to prevent expansion of one particlebed when the filling device is removed. Further variants are describedin JP-A-4-98156, JP-A-2-134562 and GB-A-2,238,257.

All of these earlier proposed solutions involve complicated technicalconstructions and/or only provide partial compensation for the resultantstability without being able to re-establish the original degree ofcompaction and density of the packed particle bed.

Accordingly, an object of the present invention is to provide a columnhaving an inlet part which, while being structurally simple, enables theoriginal compactness/density of the particle bed to be maintained, so asto avoid completely those problems that are associated with a drop inpressure in the column bed and expansion of the column bed subsequent tofilling the column. Another object of the invention is to provide amethod for filling a column in accordance with this concept.

These and other objects of the invention and advantages affordedthereby, as made apparent in the following description, are achievedwith a method and a column arrangement having the characteristicfeatures set forth in respective claims 1 and 2 and described in moredetail herebelow. Preferred embodiments of the invention are set forthin the depending claims.

The invention will now be described in more detail with reference tosome particular exemplifying embodiments of a separation columnconstructed in accordance with the invention, and also with reference tothe accompanying drawings, in which

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a longitudinal-sectioned view of one embodiment of theseparation column with the inlet part shown in a column fillingposition;

FIG. 2 is a view corresponding to the view of FIG. 1, but shows thecolumn inlet element in a locked position after completion of a fillingoperation;

FIG. 3 is a cross-sectional view taken on the line A--A in FIG. 1;

FIG. 4 is a partial longitudinal-sectioned view of another embodiment ofthe separation column and shows the column in a filling position; and

FIG. 5 is a view corresponding to the view of FIG. 4, but with the inletpart in a locked position after filling of the column has beencompleted.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The separation column illustrated in the drawings is comprised of acolumn tube 1, for instance made of glass, PEEK (apolyether-ether-ketone sold by ICI) or like material, and an inlet meansin the form of a top element 2, made for instance of PEEK. The inletmeans can be moved axially along the tube 1 and locked thereto, as willbe described in the following. The bottom part of the tube 1 includes anoutlet passage 3 which opens into a connecting recess or aperture 4which is screw-threaded for instance (not shown). A bottom filter 5 issealingly mounted on top of the outlet passage 3.

Similar to the bottom part of the tube, the top element 2 also has aconnecting recess or aperture 6 (for instance screw-threaded) whichincludes an inlet passage 7 which opens into a central recess 8 in thebottom part of the top element 2. The inner diameter of the recess 8corresponds to the outer diameter of the column tube 1, so that the topelement 2 can be moved vertically, i.e. axially, in relation to thetube 1. A resilient seal 9, which in the illustrated case is an O-ringmade of silicone rubber for instance, is received in an annular groove10 provided in the bottom of the recess 8. The recess 8 is providedinwardly of the sealing groove 10 with a circular recessed-part 11 whichis adapted to accommodate a top filter 12, made for instance of sinteredPEEK. For reasons that will be readily apparent from the followingdescription, the recessed-part 11 has a depth which is slightly smallerthan the thickness of the filter 12. Furthermore, the diameter of therecessed-part 11 in relation to the filter is such as to enable thefilter to be fixated in any desired position in the recessed-part byvirtue of a force-fit, and so that the filter can only be movedvertically in the recessed-part by applying to the filter a force whichis greater than the force to which the filter is subjected duringfilling of the column. Arranged around the periphery of therecessed-part 11 are a number, in the illustrated case six, radial slotsor channels 13 which connect the bottom of the recessed-part 11 with thespace below the sealing groove 10, and therewith also with the columntube interior 14. As will be readily understood from the followingdescription of the manner in which the column works, the configurationof these channels can be varied within wide limits, for instance withregard to both number, peripheral extension and radial depth.

When filling the column tube 1 with packing material, the top element 2will have the position shown in FIG. 1, in which it rests against theuppermost part of the tube 1 via the sealing ring 9. The filter 12 isplaced in the recessed-part 11 so as to leave a gap 15 between thebottom of said recessed-part and the upper side of the filter, and alsoso as to form a gap between the upper edge of the tube and the undersideof the filter. This enables the space 15 above the filter 11 tocommunicate with the column tube interior 14 through the channels 13 inthe side wall of the recessed-part 11.

A slurry comprising the desired particles of separation medium is pumpedinto the inlet passage 7 in per se conventional manner through aflexible tube connected to the connection 6. Because of the presence ofthe channels 3, the slurry will pass along the side of the filter 11 anddown into the column tube 1 and will continue to the outlet passage 3 inthe tube bottom. The particles are retained at the bottom of the tube bythe bottom filter 5, while liquid will pass essentially unimpededthrough the filter. The sealing ring 9 prevents liquid/slurry fromleaking along the contacting surfaces between the top element 2 and thecolumn tube 1. Eventually, the interior 14 of the tube will have beenfilled with homogeneously packed particles (not shown). In the case ofthe illustrated embodiment, this may be seen as the pumping ceasing, bythe particles clogging the channels 13 and stopping the delivery ofslurry. Naturally, it is also possible to use a suitable indicator whichwill indicate when filling of the tube is complete, for instance asuitably positioned optical sensor which detects when the particlesbegin to pack at the connection 6, or a pressure sensor which detectsthe change in pressure that occurs when the channels 13 become blockedor clogged, etc.

When the column tube is full and pumping has been stopped, the topelement 2 is pressed down over the column tube 1 to the position shownin FIG. 2, either manually or mechanically, and is firmly locked in thisposition. In this regard, the top filter 12 is compressed slightlybetween the top element 2 and the upper edge of the tube, so that thefilter abutment surfaces will seal effectively and close the earlieropen communication of the channels 13 with the column tube interior. Thesealing ring 9 will have been compressed in the groove 10 at the sametime, so as to ensure effective sealing against leakage when the packedcolumn is used. The filling arrangement is then disconnected and thepacked separation column is ready for use. It will be apparent that theparticle bed will have the same degree of compaction as that duringfilling of the column tube, in other words that the stability of theparticle bed achieved while filling the column tube will be retained andthat this stability can be maintained even at high liquid pressures,such as in HPLC.

The top element 2 can be locked in the position shown in FIG. 2 in anumber of different ways. For instance, the top element 2 may beconstructed for screw-engagement with the upper part of the column tube1, in which case the top element is brought to its sealing position byscrewing said element down along the column tube. Alternatively, the topelement may be provided with an internal (e.g. circular) recess whichcoacts with a stop shoulder (e.g. also circular) on the outside of thecolumn tube, or vice versa. In this case, there may be provided abovesuch a stop shoulder a further stop shoulder which functions to lock thetop element temporarily in the filling position illustrated in FIG. 1.The use of cotter-pins or like devices is a further alternative.

In order to ensure that the top element 2 will be held in its sealingposition (FIG. 2) in use, at least when using the stop shoulder(shoulders) alternative, it may be convenient to place a locking sleeveor the like over the top element such as to fix the element against thecolumn tube, by preventing radial expansion. It is, of course, alsoconceivable for the top element itself to lack a locking function and tolock the element in its sealing position totally with the aid of anadditional element instead, such as a sleeve-like element of theaforesaid kind or a similar sleeve-like element which can be placed overthe top element and which includes means for locking coaction with theseparation column.

Naturally, the material in the top filter 12 will need to have at leasta certain degree of rigidity, in order to achieve the intended function.If the filter material used is not sufficiently rigid in this regard,appropriate supportive means may be used, for instance the filter may beplaced in a cassette or like supportive device.

In order to ensure that the top filter 12 will not be pressed downagainst the column tube 1 by the pressure of the liquid when the topfilter 12 is in its filling position (FIG. 1), and therewith prevent thepassage of slurry past the filter, small spacer elements in the form ofpegs or the like, for instance, may be provided on the filter surfacethat abuts the column tube. These pegs, or like projections, willpreferably be configured to pierce the filter and/or be deformed to asufficient extent as the filter is pressed down onto the column tubewhen locking the top element (FIG. 2). Such spacer elements may replacetotally the frictional engagement of the filter with the side-wall ofthe recessed-part 11.

Although not specifically shown in FIG. 2, it will be understood that,in practice, the sealing ring 9 will be pressed against the partitionwall 11a between the groove 10 and the channels 13 as the top element 2is pressed down into its locked position, so as to deform the partitionwall and press said wall against the filter 12, at least to asubstantial extent. This will reduce or even essentially completelyeliminate the pocket or dead volume that is formed between the channelwall and the filter in the FIG. 2 illustration.

As will be understood, in order to prevent slurry from entering thesealing groove 10 during the filling process, the inner wall 11a of therecessed-part 11 may be extended right down to the column tube 1 in thefilling position, and then be deformed when the top element 2 is broughtto its locked state. The bottom part of the wall may optionally extendradially inwards beneath the filter to some extent. Alternatively, thepartition wall 11a may extend down into a corresponding groove in theupper side of the column tube 1 and therewith effectively prevent accessto the sealing groove.

In the case of the FIG. 2 illustration, when the filter 12 is in alocked state the filter will lie against the abutment surfaces of boththe top element 2 and the column tube 1. As will be understood, it maybe sufficient for the top filter 12 to seal solely against one of theseabutment surfaces. If desired, means may be provided for holding thefilter 12 spaced from each of these surfaces, for instance spacerelements in the bottom of the recess-part 11, so as to ensure theprovision of a liquid distributing space above the filter 12. Similarly,the aforesaid spacer elements on the column tube abutment surface neednot necessarily be configured for piercing of the filter 12, providedthat an effective seal can be achieved against the bottom-part of therecessed-part 11. Furthermore, it is not necessary for the filter 12 tobe movable in relation to the top element, but may be fixed thereinthrough the medium of spacer means.

FIGS. 4 and 5 illustrate a slightly modified embodiment of theinvention. This embodiment includes a column tube 16 which is providedwith an end-piece 17 having a larger inner diameter than that of thecolumn tube. A top element 18 can be screwed onto the end piece 17 andhas a connection 19 which is joined to an inlet passage 20. The inletpassage opens into a recessed-part 21 in the bottom of the top element18 and is adapted to accommodate a top filter 22 provided with aperipheral sealing ring 23 (for instance pressed therein). A number ofradial slots or apertures 24, for instance four in number, whichfunction as bypass channels are provided in the side-edge of therecessed-part 21 that lies proximal to the column tube 16.

FIG. 4 shows the top element 18 in the filling state of the column,wherein the top filter 22 (similar to the filter 12 in the FIG. 2embodiment) is in frictional engagement with the side-wall of therecessed-part 21 at a distance from the bottom of said recessed-partsuch that the channels 24 connect the space above the filter 22 with thespace beneath the filter, and therewith with the interior of the columntube 16.

When the filter 22 is placed in the tube-filling or tube-packingposition and the bypass channels 24 are thus open, as illustrated inFIG. 4, particle slurry is pumped into the column. When the column hasbeen filled with packed particles, the top element 18 is screwed down tothe bottom position shown in FIG. 5, while maintaining the same packingpressure. The filter 22 and the sealing ring 23 are therewith forcedpast the channels 24 and in towards the bottom of the recessed-part 21of the top element, thereby closing the channels 24. In this state ofthe arrangement, the sealing ring 23 will thus seal between the columntube 16 and the top element 18, so that liquid can only pass through thefilter 22.

In both of the aforedescribed embodiments, the bypass channels aredelimited between the top filter and recesses in the top element.Naturally, the channels may be formed instead by peripheral recesses inthe filter. Referring back to FIGS. 1 and 2, it is also conceivable toomit the partition wall 11a between the sealing groove 10 and the filteraccommodating recessed-part 11 and allow the whole of the seal 9 toextend in towards the filter 12. This enables the channels to be formedin the seal 9 itself, these channels being blocked or eliminated bydeformation as the filter is compressed when the top element is pressedto its locked position. Naturally, different combinations of thesealternatives are also conceivable.

It will be obvious that the construction of the separation column, andthen particularly its inlet part, can be varied in a number of differentways. The invention is therefore not limited in any way to theexemplifying embodiments specifically described above and illustrated inthe drawings, since the invention will, on the contrary, include allvariants that lie within the scope of the general inventive concept asdefined in the following claims.

We claim:
 1. A method of filling a liquid chromatographic column withparticulate separation medium, which comprises: introducing a liquidsuspension of the particulate separation medium into one end of thecolumn, said column being closed at one end by a first filter so thatthe particles will be retained by the filter while the liquid passestherethrough; andclosing the other end of the column with a secondfilter when filling of the column is terminated, characterized bydelivering the particle suspension through an inlet element which isdisplaceable axially in relation to the end of the column and in whichinlet element the second filter is mounted; maintaining the inletelement and the filter during the filling process in an position inwhich the particle suspension is able to pass from the upper side of thefilter past the side-edge of the filter to the interior of the column;and after completion of the filling process, displacing the inletelement towards the end of the column, while maintaining liquidpressure, to a position in which passage past the filter is prevented,and fixing the inlet element in this position.
 2. A liquidchromatographic column which is intended to be filled with particulateseparation medium and which includes a column tube having an inlet partprovided with a top filter and an outlet part provided with a bottomfilter, characterized in that the top filter is disposed in an inletelement which is displaceable axially in relation to the end of thecolumn between a filling position, in which particle suspension is ableto pass from the upper side of the top filter and flow past theside-edge of said filter to the interior of the column, and a lockableuse-position in which passage past the filter is prevented.
 3. A columnaccording to claim 2, characterized in that the top filter seals againstpassage past the filter when in said use position.
 4. A column accordingto claim 3, characterized in that in the locked use-position of the topfilter in the column, the top filter seals against the upper side of thecolumn tube.
 5. A column according to claim 4, characterized in that inthe locked use-position of the top filter in the column, the top filteralso seals against the bottom of the inlet element.
 6. A columnaccording to claim 2 or 3, characterized in that the inlet element has acentral recessed-part into which its inlet channel discharges and inwhich the filter can be moved axially, bypass channels extending betweenthe wall of said recessed-part and the filter.
 7. A column according toclaim 6, characterized in that said bypass channels are delimitedbetween the outer edge of the filter and radial recess in the wall ofthe recessed-part.
 8. A column according to claim 6, characterized inthat the bypass channels are provided at least partially in the filter.9. A column according to claim 6, characterized in that the bypasschannels are provided at least partially in the sealing element.
 10. Acolumn according to claim 6, characterized in that the bypass channelsextend along essentially the full depth of the recessed-part.
 11. Acolumn according to claim 2, characterized in that the column includes asealing element which is located radially outside the filter and whichfunctions to seal between the inlet element and the column tube.
 12. Acolumn according to claim 2, characterized in that the filter iscompressible and has a thickness which exceeds the depth of therecessed-part.
 13. A column according to claim 2 that has been filledwith a separation medium by a method of filling a liquid chromatographiccolumn with particulate separation medium, by introducing a liquidsuspension of the particulate separation medium into one end of thecolumn, said column being closed at one end by a first filter so thatthe particles will be retained by the filter while the liquid passestherethrough; andclosing the other end of the column with a secondfilter when filling of the column is terminated, characterized bydelivering the particle suspension through an inlet element which isdisplaceable axially in relation to the end of the column and in whichinlet element the second filter is mounted; maintaining the inletelement and the filter during the filling process in an position inwhich the particle suspension is able to pass from the upper side of thefilter past the side-edge of the filter to the interior of the column;and after completion of the filling process, displacing the inletelement towards the end of the column, while maintaining liquidpressure, to a position in which passage past the filter is prevented,and fixing the inlet element in this position.